The goal of this project is to determine how interactions between active site amino acid residues and various ligand moieties contribute to enzyme specificity and affinity. An understanding of such interactions is pivotal to the design of therapeutic drugs targeted towards proteins of known structure, to the design of novel catalysts by engineering enzymes, to an understanding of catalysis, and to an understanding protein evolution. This proposal describes two general approaches for producing enzymes with changed specificities. One approach uses site directed mutagenesis to replace specific sequences and super secondary structures with those of related enzymes. A second approach mimics the processes of long-term adaptive evolution by placing constructed strains of E. coli under the intense selective pressures imposed by chemostat culture. These approaches, either independently or in combination, will yield enzymes with altered specificities. Site directed mutagenesis of evolved/mutated enzymes and kinetic studies using substrates and substrate analogues, will then be employed to determine the causes of changes in specificity. The use of natural selection to produce enzymes of changed specificity is particularly attractive because no a priori decisions regarding which residues to target for study are necessary. Hence the chances of discovering unforeseen determinants of specificity are maximized. Also, mutations conferring differences in growth rates as small as 1.0% per generation, not detectable using selection on petri plates, are readily detectable in chemostat culture. Thus, chemostat competition experiments provide a means to evolve specificity by small increments. The isocitrate dehydrogenase (IDH) of Escherichia coli and the related isopropylmalate dehydrogenase (IMDH) will be used as a model system. These enzymes catalyze an oxidative decarboxylation reaction at the 2R- malate core common to their substrates. Interactions between active site residues of each enzyme and the various gamma-moieties of the substrates, which are attached at the 3S position of the common core, provide an obvious means to generate specificity. High resolution structures of native and phosphorylated IDH, and of the binary complexes with isocitrate and with NADP, are available. Also available is a high resolution structure of IMDH from Thermus thermophilus. These, together with our understanding of the kinetic and catalytic mechanisms and the means by which phosphorylation regulates IDH activity, provide the basic information necessary for detailed interpretations of structure-function relations. The development of new general approaches to obtain answers to specific ligand-protein binding problems, and without regard for the preconceived notions of the experimentalist, is crucial to discovering how otherwise unforeseen determinants influence the affinities and specificities of proteins. The development of such approaches will also provide greater insights necessary for the rational design of drugs and novel biological catalysts, and an understanding of catalysis and protein evolution.